The overall goal of this protocol is to combine immunofluorescence staining with DNA fluorescent in situ hybridization on a 3D functionalized wire and to identify immunophenotype and FISH signals on the support using a conventional widefield fluorescence microscope. This method can help answer key questions in the field of secreting tumor cell characterization such as identifying therapy related targets on circulating non-small-cell lung cancer cells. The main advantage of this protocol is allowing the simultaneous identification of phenotypic parameters such as EpCAM positivity and cytosomatic alterations such as ARC gene status to detect putative CTCs.
Although this method was uniquely designed to provide in situ insulin cancer CTC features, it can also be applied to the study of other cancer like breast cancer. We developed this protocol while handling the wire for direct immunophenotyping and CTC detection. To obtain more data, we associated an immunophenotyping protocol with a DNA fluorescence in situ hybridization protocol.
Handling the wire is not easy, mainly because the functionalized tip is fragile. A visual demonstration will have the readers reproducing the steps. To begin the experiment, resuspend the entire contents of a T75 flask that is at 80%confluence in a five milliliter vial using four milliliters of complete medium.
Next, remove the wire from its glass packaging. Dip the functionalized gold part of the wire into the vial, ensuring that the wire stopper is a watertight fit for a five milliliter vial. Seal the wire with laboratory film.
Then, incubate the wire in a tube rotator for 30 minutes at room temperature. After the rotation, rinse the wire three times in one-fold PBS solution using three different clean vials and wait for the wire to dry. After air drying the wire for 10 minutes in a hood, fix the cells by dipping the wire in 100%acetone solution in a five milliliter tube for 10 minutes at room temperature.
Ensure there is no trace of acetone on the functionalized tip. Air dry the wire for five minutes at room temperature. Next, in a five milliliter tube, wash the wire twice in one-fold PBS.
Incubate the wire in antibody dilution buffer. Prepare 150 microliters of antibody mix with a dilution of monoclonal primary antibody conjugated with fluorochrome and antibody dilution buffer specified in the datasheet. Then, use a P200 tip to extract the wire from the wire stopper and gently insert the wire through the larger hole of the tip.
Insert the unfunctionalized end first and slowly pull the wire through the smaller hole until the gold part is just beyond the larger hole. Gently add 150 microliters of the antibody mix into the tip. To avoid the risk of bubble formation, gently twirl the wire until the functionalized end is completely plunged.
Seal the hole of the tip and wrap laboratory film around the hole. Incubate the wire vertically overnight in the dark at four degrees Celsius. On the second day, block the antibody incubation by washing the wire twice in one-fold PBS.
Reinsert the wire in its wire stopper. Three hours before the DNA-FISH protocol, set the hybridization oven to 75 degrees Celsius and set the dry heat oven at 37 degrees Celsius. Cool the 0.4-fold SSC solution to four degrees Celsius.
Wash the wire three times in ice-cold 0.4-fold SSC solution. Dry the wire in the dark under the fume hood cupboard for 10 minutes. After 10 minutes, vortex and spin the probe for five seconds.
Drop 10 microliters of the probe into glass microtubes, then cover them with laboratory film. Wrap the microtubes in dry absorbent paper. Place into a 50 milliliter tube and spin them briefly.
Carefully place the dried wire into the microtube and insert the wire stopper. Then, seal the wire stopper with rubber cement. Place the wire into a dark humid chamber in the hybridization oven for eight minutes at 75 degrees Celsius to obtain complete DNA denaturation.
After eight minutes, move the humid chamber into a dry heat oven at 37 degrees Celsius overnight. Place the slide staining jar with 0.4-fold SSC solution into a water bath set at 72 degrees Celsius. Check the 0.4-fold SSC solution temperature until it reaches 72 plus or minus one degree Celsius.
Next, prepare two glass beakers. One with two-fold SSC plus 0.05%Tween solution and the second one with distilled water. Remove the humid chamber for the dry heat oven.
Use tweezers to carefully remove the rubber cement from the wire stopper, and pull the uncoated end of the wire through the end stopper, being careful not to damage the functionalized tip. Dip the wire into the 0.4-fold SSC solution for two minutes at 72 degrees Celsius. Wash the wire in two-fold SSC plus 0.05%Tween solution for 30 seconds at room temperature.
Then, wash the wire in distilled water at room temperature. Dry the wire under the fume hood for 10 minutes in the dark. Incubate the functionalized tip of the wire with previously prepared DAPI solution in a two milliliter vial.
Rinse the wire twice in one-fold PBS and air dry the wire. Position the wire in the wire holder and carefully insert the functionalized tip through the entry point of the special holder until the tip matches the coupling point. Place the special holder on the microscope stage.
Adjust the focus of the microscope using a 20-fold lens. First, coarsely focus on the special support and then adjust finely on cells that appear bright in the DAPI channel. Only focus the cells central to the lens and acquire images using a 12-bit digital camera in 20x and 40x objectives with appropriate filters for red, green and blue probes.
Finally, prepare the wire for long-term storage by packing the wire next to the functionalized tip and carefully insert the wire into a glass tube. Store the wire vertically or horizontally at negative 20 degrees Celsius. Immunofluorescence staining did not interfere with DNA-FISH signals.
When the immuno-DNA-FISH assay was performed on the 3D support functionalized wire, EpCam staining was clearly visible. The ALK probe revealed the gene deletion. NCI-H3122 cell line showed an aberrant LAK-gene status in contrast to the NCI-H1975 with a wild type ALKG.
Once mastered, this protocol can be done in nine total working hours over three days. While attempting this procedure, it's important not to touch the functionalized part of the wire to prevent any damage. Different antibodies and FISH probes can be used following this protocol to answer additional questions such as identifying cells with other phenotypic markers and other cytogenetic alterations such as NAT or HAR2 gene amplification.
After watching this video, you should have a good understanding of how to identify simultaneously putative CTCs through phenotypic parameters such as EpCAM positivity and to detect cytogenetic alterations such as ARC gene status. Don't forget that working with acetone and oven can be extremely hazardous. Precautions such as handle acetone in hood and wearing protective gloves should always be taken while performing this procedure.
